Breathing is generated by a neuronal network that is distributed along the rostro-caudal axis of the ventrolateral medulla (ventral respiratory column, VRC). This network gives rise to three distinct phases: inspiration (I), post-inspiration (Post-I), and active expiration (AE). Many disorders are associated with disturbances in different forms of breathing and the response to hypoxia. Thus understanding how these phases of breathing are generated and dynamically regulated under various conditions such as hypoxia is of great basic scientific and clinical interest. In this project we introduce two novel rhythmicall active brainstem slice preparations that allow us to study the integration of synaptic, intrinsic and modulatory properties in the wider medullary network to an extent that was not possible before. Based on our preliminary data we propose the hypothesis that post-I and inspiration are generated by an excitatory column that extends rostrally from the pre-Btzinger complex into the Btzinger complex. This distributed excitatory network interacts with GABAergic and glycinergic mechanisms as well as intrinsic membrane properties that will be explored in the horizontal slice preparation using a variety of electrophysiological, pharmacological, and optogenetic approaches (Aim 1). Insights gained in this horizontal slice preparation will be compared with data obtained from two transverse slice preparations that isolate the caudal and rostral portion of this excitatory column (Aim 2). This approach will allow us to differentiate rhythmogenic mechanisms occurring at the caudal and rostral end of this column. Concepts revealed in these in vitro findings will then be tested in a spontaneously breathing in vivo preparation (Aim 3). We expect that the introduction of these novel in vitro preparations, combined with modern optogenetic techniques and a rigorous integration with in vivo approaches will allow us to revisit existing models of respiratory rhythm generation. This may lead to a better understanding of various issues that remain unresolved and unexplained at the current state of knowledge in the field of neural control of breathing.